This investigation proposes to delineate two critical elements in the ecology of Eastern equine encephalitis (EEE) virus: (1) the molecular variation exhibited by EEE viruses over time within several different geographic foci of infection, and (2) elucidation of the overwintering maintenance mechanism(s) through the novel approach of using molecular probes. EEE virus is a mosquito-borne alphavirus which is widely distributed throughout both North and South America. Disease occurs annually. Although major outbreaks are intermittent, infection results in serious disease with high fatality and significant sequelae in survivors. Much work has been done to describe the epidemiology of EEE virus, but many questions remain unanswered concerning the ecology of EEE viruses, e.g., the number of different EEE strains circulating in a focus at any one time, the genetic and antigenic variation between endemic and epidemic strains, and the vector(s) responsible for transmitting disease to equines and humans. Similarly, although many hypotheses exist for the overwintering maintenance mechanism(s) of EEE virus, all previous attempts to isolate infectious virus in order to delineate the insect or mammalian winter reservoir(s) have been unsuccessful. In the studies outlined, a combination of molecular methods will be used to identify and characterize EEE viruses isolated from several different geographic foci over time. Characterization of viral RNA by oligonucleotide fingerprinting, hybridization, and restriction fragment analysis of virus-specific cDNA, and analysis of viral proteins by heterologous immunoprecipitation and enzyme digest analyses will enable us to define the degree of genetic and/or phenotypic differences occurring among viruses circulating in several natural endemic foci. Using this information, molecular probes will be developed for specific and rapid identification purposes. Finally, these probes will be used to directly examine tissues of suspected vertebrate hosts and invertebrate vectors for the presence of latent or persistent virus infections, in order to define to elusive overwintering maintenance mechanism(s) of EEE viruses. Once these aspects of EEE virus ecology have been determined, it may be possible to implement more effective surveillance and control strategies to curb outbreaks of this debilitating and highly fatal disease. This information may also serve as a model for defining the ecology of other important arboviruses.